遺伝子検出による迅速微生物解析技術の開発
Contents
1. 2 PCR E vulneris E amnigenus H alvei y TU 55 J niu LAM E E QNSE S 300 s LANES NER SENE IRR CERA OMERE 5 S MN PA EN OREWA 7 O6 e A LA RR NER ARIS QUEMAR 109 59 57 m 10 ES PCR Ig E RUE E dit lr eo t on WERE X3 A PCR Klebsiella pneumoniae IAM 1063 Klebsiella oxytoca ATCC 43086 O pem sa RU ke us em FR Rs nd paeem Pe Ma RO P EDEN pov m Rm sS eap T Citrobacter feundii NBRC13546 Enterobacter aerogenes NBRC12010 ea Sd RAEE eg na sv P m Leclercia adecarbexylata ATCC23216 PESE ed PR L ama zc PNE E ied Raoultella planti2. HECU AA 367 T FvEKBEI UATUT 0157 PCR Polymerase Chain Reaction PCR PCR PCR 3 2 2 1 1 Ig IRE SE TES EF WERE mois 2011 LB 35C 20 24 2 2 I ISOPLANT I MagPrep Bacterial Genomic DNA Kit Novagen
3. BOI 2011 Buc 1 2 3 4 5 6 7 8 4 2006 72 New Food Industry 52 2010 1 Hein I Lehner A Rieck P Klein K Brandl E and Wagner M Comparison of different approaches to quantify Staphylococcus aureus cells by real time quantitative PCR and application of this technique for examination of cheese Appl Environ Microbiol 67 2001 3122 Martinez Blanch J F Sanchez G Garay E and Aznar R Development of a real time PCR assay for detection and quantification of enterotoxigenic members of Bacillus cereus group in food samples Int J Food Microbiol 135 2009 15 Nam H M Srinivasan V Gillespie B E Murinda S E and Oliver S P Application of SYBR green real time PCR assay for specific detection of Salmonella spp in dairy farm environmental samples Int J Food Microbiol 102 2005 161 Omiccioli E Amagliani G Brandi G
4. 1 TII 5 E LC E vulneris E amnigenus H alvei 1F IR PCR 2 E vulneris E amnigenus PCR EADIE H alvei PCR vulneris PCR 12 11 PCR 3
5. 50 ul L LXTEGX 0 2 mM dNTP i 2 5U ExTaq DNA polymerase tb 0 5 uM Forward Reverse IF IR PCR 94 5 94 1 2f 55 1 30 72 1 30 72 CT 5 ZL 1675 ZR 2025 PCR 94 C 5 94 1 60 1 Zeus 22 1 1 KC 25 72 52x 99 2 4 PCR PCR OKIDI WREX 50 ul iQ SYBR Green Supermix Bio Rad 25 ul 05 uM 1F IR PCR 1E 1R PCR 2 5 10 g 90 ml 1 6 X10 cfu 1 6X10 cfu 1 6X 10 cfu E amnig
6. 10 PCR 2 30 3 24 1 8 1 30 300 cfu 2 PCR 1 1X10 cfu ml 1 1 X 10 cfu ml 4 1 51 PCR 48 94 Q 1 5 3 PCR 10 I0 cfu ml 4 10 cfu g PCR 2 30 8r og
7. 2 1F 1R Ig IR PES di rie trt cl Ii PIA 2011 1 PCR Lo dup e ee 1F ZL 1675 1R ZR 2025 Buttiauxella agrestis sus JCM 1090 T po M Citrobacter amalonaticus ICM 1661 T od 0 3 Citrobacter amalondticus NBRC 13547 to Citrobacter bradki ATCC43162 5 oL Citrobacter frewdu NBRC13339 ol Citrobacter fewdi NBRC 13546 Citrobacter eo NBRC16624 Eon Re M Dr pite itt E M pe e pemet M o Sede e CN NNNM MD ANM cora NNI T NM Eee E M C ME NINE Er NN NE Mr DT S A ML CEN T Eee e dM EU M ee M E LM a M Zmote cows impsum WAMDM frere cac tsp cto I ee L1 pue ee Eee M Stet NN espe A exe con NN S NN RR Perche loc m S El E CSLNNENUNE T ANE TRNEM Klebsiella oxytoca ATCC824 Klebsiella oxytoca ATCC43086 Klebsiella oxytoca ATCC 43165 toD Klebsiella oxytoca ATCCTWSM o Klebsiella oxytoca ATcc4931 Klebsiella oxytoca ATCC43863 Klebsiella pneumoniae IAM 1063 OD Klebsiella pneumoniae NBRC 3318 d NN Klebsiella pneumoniae NBRC3319 NN Klebsiella pneumoniae NBRC3321 Klebsiella pneumoniae NBRC3512 Klebsiella pneumoniae NBR
8. DNA TERR US ic 2 D f D vc Bi Hs ze 80C C 8 XD U 7 R SCT 3 250ml B PER Bacterial Protein Extraction Reagent Thermo Fisher Scientific 50ml 2 13 000 rpm 2 500 W 2 13 000 rpm 2 50ul UltraClean PCR Clean up DNA Purification Kit MO BIO Laboratories 2 3 PCR 1E CTG GAA GAY CAG GAY ATG TGG CGS ATG AGC GG 3 1R S CAG CGC ACG GCG TTR AAR YTG TKC TGC TTC AT 3 C5 HELL C HI TES ESLEERR HH 77 R 7 3 ZL 1675 5 ATG AAA GCT GGC TAC AGG AAG GCC 3 ZR 2025 5 GGT TTA TGC AGC AAC GAG ACG TCA 3 PCR
9. PCR 99 E vulneris RA E E amnigenus 55 dei 33 JO E vulneris PCR 2 30 1 6X10 1 6 x 10 cfu g PCR 1 610 cfu g PLANS Se Y E amnigenus lacZ 3 Ig E RUE E dit lr eo t cn WERE PCR Base Line Subtracted CF RFU 20 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 Cycle 3 C E amnigenus 1 6 10 cfu C E amnigenus 1 6 10 cfu C E amnigenus 1 6 10 cfu C E amnigenus 10g 10g Nr 10g 10g AONO 20
10. PCR 1 MAC UR Rapicom 2 MAC 4 l
11. 7000 7000 6000 6000 5000 5000 4000 4000 3000 3000 2000 2000 PCR Base Line Subtracted CF RFU 1000 1000 1000 1000 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 M 36 38 40 42 Cycle 1 PCR 5 5X 10 cfu ml E amnigenus 1 1 X10 cfu 10 10 PCR BIA 2011 M 1 Wr ag 397 d OQ dg wwe 2 PCR M 100bp 10 1 1 107 cfu ml 1 1X 10 cfu m T 3 4 E amnigenus PCR 3 AE E amnigenus PCR E amnigenus PCR Ct 0 996
12. and Magnani M A new platform for Real Time PCR detection of Salmonella spp Listeria monocytogenes and Escherichia coli O157 in milk Food Microbiol 26 2009 615 Sails A D Fox A J Bolton F J Wareing D R and Greenway D L A real time PCR assay for the detection of Campylobacter jejuni in foods after enrichment culture Appl Environ Microbiol 69 2003 1383 Bej A K McCarty S C and Atlas R M GEREZA Z MERE Detection of coliform bacteria and Escherichia coli by multiplex polymerase chain reaction comparison with defined substrate and plating methods for water quality monitoring Appl Environ Microbiol 57 1991 2429 9 Bej A K Steffan R J DiCesare J Haff L and Atlas R M Detection of coliform bacteria in water by polymerase chain reaction and gene probes Appl Environ Microbiol 56 1990 307 10 2004 BOIA 2011
13. CINSS Klebsiella pneumoniae IAM 12351 O Klebsiella pneumoniae NBRCINSS feme remit S elem Koea mee amu rlo E E a e Pus D A emea I AME le M p S arome E A EV I AM NEN EUN re aerial I RN ANE ME C MM Eee Ramila paote TN E eee E E m CMM HN NN T NN A gee femteat Eun M WE PCR IAM Institute of Applied Microbiology JCM Japan Collection of Microorganisms NBRC NITE Biological Resource Center ATCC American Type Culture Collection 51 PCR 48 4 l1 XPREOCKHSEHBEISEHIHZ 74 ZL 1675 ZR 2025 PCR 14 27 8 2 1E71R PCR PCR 3 2
14. Ig REGE TESI EF WERE PIA 2011 CES R Development of Rapid Microorganism Analysis System Based on the Gene Detection TOMINAGA Tatsuya SEKINE Masahiro PCR zcZ PCR 1 48 1 5 PCR XE PCR Ct 0 99 PCR 3 1 8
15. cola JCM SV E LS mc 1 3 1E71R PCR PCR 1 PCR PCR 10 f PCR CO LLT M O 0 991 PCR 88 10 10 Ct PCR 2 10 10 PCR amnigenus lacZ 9000 9000 8000 8000
16. enus 1m1l 100 1 10 1 30 PHRA UI RUE 1 ml PCR AREL o IEEE Cv 3 3 1 O lacz THV ERE KA lacz Citrobacter koseri Escherichia coli Cronobacter sakazakii Klebsiella pneumoniae Enterobacter cloacae lacZ 3 lacz
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